Validation of sex determination genes in the Coho salmon (O. kisutch) genome and development of a rapid diagnostic test using CRISPR/Cas12

Sex determination in Coho salmon (Oncorhynchus kisutch) is an essential issue for aquaculture and ecological assessment but is challenging due to late-emerging sexual dimorphism. This study tackles the need for early, non-lethal sex identification by combining genomic analysis with an innovative CRISPR diagnostic tool. Using genome-wide association studies (GWAS) and whole-genome sequencing (WGS) across diverse populations, we validated the male-specific indel at the pseudogene GH2 on chromosome 10. Following Mendelian segregation of heterozygous males and homozygous females, we identified a novel sex-associated locus on chromosome 30 linked to E3 ubiquitin ligases. Strikingly, the SNPs in this gene follow the same segregation pattern of the deletion in chromosome 10, suggesting a founding event following a speciation event that occurred in O. kisutch. Another possible explanation is the ancestral tetraploidy of the genera, but in this case, both regions do not appear to be paralogous. This pattern has been observed in other members of the Salmonidae family. The transcriptome analysis of immature gonads revealed sex-specific gene expression in several genes on chromosome 30, but little overlap with the GWAS. We developed a CRISPR/Cas12-based diagnostic test targeting the GH2 indel, achieving 100 % accuracy in sex determination with a detection limit of 0.1 ng/μL of DNA. Requiring minimal equipment, this field-ready assay outperforms traditional methods like high-resolution melting (HRM) analysis in speed and portability. Our results will help deepen the understanding of sex determination in salmonids and provide a practical, scalable tool for aquaculture and ecological applications.